EliKine Free Thyroxine (ft4) ELISA Kit Booklet Item NO. KET0005 Product Name EliKine Free Thyroxine (ft4) ELISA Kit ATTENTION For laboratory research use only. Not for clinical or diagnostic use
TABLE OF CONTENTS INTRODUCTION Background... 1 Assay principles... 1 Assay restrictions... 1 PRODUCT INFORMATION Materials supplied... 2 Other supplies required... 2 Storage conditions... 2 Precautions... 3 Technical hints... 3 ASSAY PROTOCOL Sample collection & storage... 4 Reagent preparation... 4 Assay procedure... 4 DATA ANALYSIS Calculation of results... 6 Typical data... 6 Precision... 7 Recovery... 7 Sensitivity... 7 Linearity... 7 Specificity... 8 OTHER INFORMATION Assay protocol summary... 9 Plate layout... 10
INTRODUCTION Background Over 99% of Triiodothyronine (T4) circulates in blood is bound to carrier proteins; thyroxine-binding globulin (TBG). However, only the free (unbound) portion of T4 is responsible for the biological action. Further, the concentrations of the carrier proteins are altered in many clinical conditions, such as pregnancy. In normal thyroid function as the concentrations of the carrier proteins alters, the total T4 level changes so that the free T4 concentration remains constant. Thus, measurements of free T4 concentrations correlate more reliably with clinical status than total T4 levels. The increase in total T4 levels associated with pregnancy, oral contraceptives and estrogen therapy result in higher total T4 levels while the free T4 concentration remains basically unchanged. Assay principle EliKine ft4 ELISA Kit employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to ft4. Standards or samples are added to the appropriate microtiter plate wells with Biotin-conjugated ft4. A competitive inhibition reaction is launched between ft4 (Standards or samples) and Biotin-conjugated ft4 with the pre-coated antibody specific for ft4. The more amount of ft4 in samples, the less antibody bound by Biotin-conjugated ft4. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Substrate solution is added to the wells and the color develops in opposite to the amount of ft4 in the sample. The color development is stopped and the intensity of the color is measured. Assay Restrictions Do not mix or substitute reagents with those from other lots or sources. It is important that the calibrator diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate calibrator diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by other factors present in biological samples. Until all factors have been tested in the ELISA Immunoassay, the possibility of interference cannot be excluded. 1
PRODUCT INFORMATION Materials supplied ft4 microplate: 96 well polystyrene microplates (12 strips of 8 wells) coated with the an antibody specific to ft4. ft4 standard: ft4 in a buffered protein base with preservatives, liquid. Biotin conjugated ft4: liquid. Avidin-HRP: liquid. HRP substrate A: Urea hydrogen peroxide solution. HRP substrate B: TMB (Tetramethyl-benzidine) solution. Stop solution: 2 mol/l sulfuric acid. Wash buffer: PBS with 0.5% Tween-20, 20 liquid. Plate covers Other supplies required Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 630 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. 500 ml graduated cylinder. Storage conditions The unopened kit should be stored at 2-8 C for 1 year. Immediately after use remaining reagents should be returned to cold storage at 4 C. Recommended storage instruction for opened/reconstituted kit components are listed below. Kit components ft4 microplate ft4 standard Biotin conjugated ft4 Avidin-HRP HRP substrate A HRP substrate B Stop solution Wash buffer (20 ) Storage conditions Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8 C. May be stored for up to 6 months at 2-8 C. May be stored for up to 1 year at 2-8 C. 2
Precautions 1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer. 2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8 C in their pouch with the desiccant provided. 3. Mix all reagents before using. Remove all kit reagents from refrigerator and allow them to reach room temperature (20-25 C). Technical hints When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. When using an automated plate washer, adding a 30 second soak period following the addition of Wash Buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. 3
ASSAY PROTOCOL Sample collection & storage The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated. Serum - Use a serum separator tube and allow samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 C. Avoid repeated freeze-thaw cycles. Plasma - Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 C. Avoid repeated freeze-thaw cycles. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples should be aliquoted and must be stored at -20 C to avoid loss of bioactive ft4. If samples are to be used within 24 hours, they may be stored at 2 to 8 C. Avoid repeated freeze-thaw cycles. Prior to assay, the frozen sample should be brought to room temperature slowly and mixed gently. Reagent preparation Bring all reagents to room temperature before use. If crystals have formed in the Buffer Concentrates, warm them gently until they completely dissolved. Wash buffer - Dilute with Distilled or deionized water 1:20. Sample dilution - If your samples need to be diluted, Deionized or distilled water is used for dilution of serum/plasma samples. Assay procedure 1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the microplate. 2. Set a Blank well without any solution. 3. Add 50 μl of Standard or Sample per well. Standard need test in duplicate. 4. Add 50 μl of Conjugate to each well (not to Blank well). Mix well and then incubate for 60 minutes at 37 C. 5. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (250 μl) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 10 seconds, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 6. Add 50 μl of avidin-hrp to each well (not to Blank well). Mix well and then incubate for 30 minutes at 37 C. 4
7. Repeat the aspiration/wash process for three times as in step 5. 8. Add 50 μl of Substrate A and 50 μl of Substrate B to each well, mix well. Incubate for 15 minutes at 37 C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 9. Add 50 μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing. 10. Determine the optical density of each well within 15 minutes, using a microplate reader set to 450 nm. 5
O.D. Value DATA ANALYSIS Calculation of results Average the duplicate readings for each standard and sample and subtract the average zero standard optical density (O.D.). Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit. As an alternative, construct a standard curve by plotting the concentration on the y-axis against the mean absorbance for each standard on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the ft4 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Typical data The standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed. Detection range: 4 pmol/l-64 pmol/l ft 4 standard (pmol/l) 6
Standard ft4 Optical Density (450 nm) Average (pmol/l) 4 1.245 1.270 1.295 8 0.651 0.6365 0.622 16 0.324 0.3045 0.285 32 0.181 0.185 0.189 64 0.093 0.094 0.095 Precision Intra-assay Precision (Precision within an assay) Four samples of known concentration were tested twenty times on one plate to assess intra-assay precision. The CV (%)<15%. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components. The CV (%)<15%. Recovery The recovery of ft4 spiked to different levels in samples throughout the range of the assay in various matrices was evaluated. The recovery ranged from 90% to 110% with an overall mean recovery of 100%. Sensitivity The minimum detectable dose (MDD) of ft4 is typically less than 4.0 pmol/l. The MDD was determined by adding two standard deviations to the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration. Linearity To assess linearity of the assay, samples containing and/or spiked with high concentrations of ft4 were diluted with the appropriate calibrator diluent to produce samples with values within the dynamic range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99. Specificity 7
EliKine ft4 ELISA Kit has high sensitivity and excellent specificity for detection of ft4. No significant cross-reactivity or interference between ft4 and analogues was observed. 8
OTHER INFORMATION Assay protocol summary 9
Plate layout 10